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PubMed1988

Photoaffinity labeling of opiate receptors using intrinsically photoactive 3H-opiates.

Opiate receptors in rat and cow brain membranes have been labeled irreversibly using the intrinsic photolability of 3H-opiates. Membranes were incubated with 3H-ligand and then irradiated with UV light of 254 nm. Nonspecific binding was determined in the presence of 10 microM unlabeled levallorphan. Irreversible binding was defined as binding which survived heat or acid denaturation of membranes. Specific incorporation of label into denatured samples was observed only when unbound or loosely bound 3H-ligand was washed free from the membranes prior to irradiation. There was a general correlation between photosensitivity of the 3H-ligand and its ability to photolabel receptors. Hence, photolabeling presumably results by covalent attachment of highly reactive species generated during photochemical decomposition of ligand. With 3H-etorphine, optimal irradiation time was 5 min. In addition to 3H-etorphine, receptors could be labeled irreversibly with 3H-oxymorphone, 3H-dihydromorphine, and 3H-ethylketocyclazocine. Of the specific binding present in irradiated, nondenatured samples, 45-60% remained attached to receptors upon denaturation. 3H-Ethylketocyclazocine exhibited an 86% yield of incorporation. Signal-to-noise levels of 50-80% could be achieved in denatured samples. Therefore, this method provides a means of covalently labeling opiate receptors in high yield and with high signal-to-noise ratios. The opioid peptides, 3-H-D-Ala2,D-Leu5-enkephalin, 3H-D-Ser2,Leu5,Thr6-enkephalin, 3H-D-Ala2,Met5-enkephalin amide, and 3H-D-Ala2,N-MePhe4,Gly-ol5-enkephalin, as well as the benzomorphan, 3H-bremazocine, apparently lack the structural characteristics which allow photolabeling. 3H-Etorphine was incorporated at high specific activity into a glycoprotein fraction of bovine brain membranes which was retained on wheat germ agglutinin affinity columns. Unretained proteins were essentially unlabeled. 3H-Etorphine was also incorporated at high specific activity into proteins of 17,000 and less than 13,700 Da and, at lower specific activity, into a protein of 2.4 X 10(6) Da in guinea pig brain membranes when kappa receptors were selectively labeled. This method therefore allows labeling of specific membrane proteins and will prove useful in molecular characterization of opiate receptors.

Affinity Labels↗
PubMed1988

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis.

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the human erythrocyte glucose transporter with 8-azidoadenosine.

8-Azidoadenosine was employed as a possible covalent probe of the erythrocyte nucleoside transporter. 8-Azidoadenosine was shown to enter human erythrocytes by a saturable mechanism (apparent Km for influx 80 microM) that was inhibited by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, and competitively inhibit uridine influx and NBMPR binding. Irradiation with UV light of human erythrocyte membranes or a partially purified preparation of the nucleoside transporter in the presence of [3H]8-azidoadenosine and dithiothreitol (as a free radical scavenger) resulted in selective covalent incorporation into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr 66,000-45,000). Covalent labeling of band 4.5 was inhibited by adenosine, uridine, and inosine, but NBMPR had no effect. Surprisingly, D-glucose and cytochalasin B, but not L-glucose and cytochalasin E, blocked covalent attachment of the ligand. No incorporation of radioactivity into membranes from rabbit and pig erythrocytes was observed, cells which transport nucleosides rapidly, but have little or no functional glucose carrier. Limited treatment with trypsin of unsealed human erythrocyte membranes photolabeled with [3H]8-azidoadenosine yielded a single radioactive fragment of Mr 19,000, a pattern identical to that obtained with [3H]cytochalasin B-labeled membranes. These results suggest that, despite 8-azidoadenosine being a permeant for the nucleoside transporter, under photoactivation 8-azidoadenosine preferentially labeled the glucose carrier.

Adenosine↗
PubMed1987

[3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter.

Irradiation of erythrocyte ghosts in the presence of [3H]forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of [3H]cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.

Affinity Labels↗
PubMed1988

Characterization of cardiac A1 adenosine receptors by ligand binding and photoaffinity labeling.

[125I]N6-(p-aminobenzyl)adenosine and [125I]N6-(p-azidobenzyl)adenosine, which are potent agonists at A1 (Ri) adenosine receptors, have been used to characterize the adenosine receptor in membranes prepared from newborn chick heart. Scatchard analyses of [125I]N6-(p-aminobenzyl)adenosine binding to cardiac membranes revealed that the ligand bound to two affinity states of the receptor with Kd values of 0.7 and 9.9 nM. The corresponding maximum binding (Bmax) values were 25 and 86 fmol/mg of protein, respectively. In the presence of 0.1 mM 5'-guanylyl imidodiphosphate, a single affinity state was detected with a Kd of 9.4 nM and a Bmax of 96 fmol/mg of protein. Direct and indirect ligand binding studies with several adenosine receptor agonists and antagonists were used to compare the characteristics of the cardiac receptor with those of the A1 receptor in the cerebellum. The binding properties of the receptors in the two tissues were very similar although marked differences were observed in the binding kinetics of [125I]N6-(p-azidobenzyl)adenosine. Photo-affinity labeling experiments followed by sodium dodecyl sulfate-gel electrophoresis showed that the cardiac receptor had a apparent molecular weight of 37,600, which was slightly but significantly higher than that of the cerebellar receptor (35,500). The present results show that the cardiac receptor has ligand binding properties and a minimal subunit molecular weight similar to the more thoroughly studied A1 receptor in neural tissue.

Adenosine↗
PubMed1986

Identification of calmodulin-binding proteins in pure mitochondria by photoaffinity labeling.

Calmodulin-binding proteins (CaM-BPs) were identified in the submitochondrial fractions obtained from highly purified rat liver mitochondria. The matrix fraction contained five CaM-BPs with apparent molecular weights (MW) of 27K, 38K, 47K, 76K, and 84K Da in a Ca solution. Electron transfer particles also contained five CaM-BPs, but their MWs were 31K, 35K, 53K, 66K, and 73K in a Ca solution. Nonspecific calcium-independent CaM-BPs were also identified in matrix fractions, having MWs of 10K, 25K, and 49K Da.

Animals↗
PubMed1985

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue.

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [3H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cation. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/3H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cation are present.

Affinity Labels↗
PubMed1985

Active site-directed photoaffinity labeling and partial characterization of oligosaccharyltransferase.

Oligosaccharyltransferase, the enzyme that catalyzes the transfer of the oligosaccharide chain of dolichol-P-P-GlcNAc2Man9Glc3 to asparagine residues in -Asn-X-Thr/Ser- sites within polypeptides, has been radiolabeled using a photoactivatable azido tripeptide acceptor, N alpha-[3H]Ac-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the oligosaccharyltransferase polypeptide from hen oviduct microsomes is 60 kDa. Radiolabeling of the 60-kDa polypeptide was completely dependent upon photolysis of hen oviduct endoplasmic reticulum preparations in the presence of the azido peptide and Mn2+, which is required for enzymatic activity. Labeling of the enzyme was not inhibited in the presence of a 10-fold excess of the nonacceptor peptides, unacetylated Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2 or unacetylated Asn-Leu-Thr-NH2, whereas it was completely abolished by the presence of a 10-fold excess of the competing acceptor peptide, N alpha-Bz-Asn-Leu-Thr-NH2. Thermal inactivation of oligosaccharyltransferase was achieved by heating endoplasmic reticulum preparations to 60 degrees C. This loss of enzyme activity at 60 degrees C paralleled a comparable decrease in radiolabeling of the 60-kDa polypeptide, whereas temperatures of 50 degrees C and lower had no effect on either process. Oligosaccharyltransferase itself may be an N-linked glycoprotein, because the 60-kDa radiolabeled polypeptide binds to concanavalin A-agarose and is susceptible to digestion by beta-endohexosaminidase H.

Affinity Labels↗
PubMed1979

Establishment of photoaffinity label derivatives of fluorene as probes in studies of chemical carcinogenesis in mammalian cell culture.

Several azido fluorenes, photosensitive analogs of the established carcinogen 2-acetylaminofluorene, have been synthesized to serve as probes in chemical carcinogenesis and mutagenesis studies. Unlike 2-acetylaminofluorene, these compounds do not require metabolic activation. However, alkylation to critical targets is achieved through the generation of nitrenes by photolysis at 360 nm in situ. We have found that the bifunctional azidofluorenes 2,5-diazidofluorene and 2,7-diazidofluorene were more toxic to and more transforming of the mouse embryo C3H 10T1/2 clone 8 cells when photolyzed in situ than were the monofunctional azides 2-azidofluorene and 7-bromo-2-azidofluorene. When the drugs were photolyzed in phosphate-buffered saline (pH 7.4) and then added to the cells these preirradiated derivatives were not transforming but were slightly toxic. However, there is not relationship between the number of photosensitive azido groups and the cytotoxicities of the preirradiated derivatives as measured by the plating efficiency method. The transformation experiments were performed under conditions in which photolysis was carried out for as little as 15 sec. This is significant because the major events that lead to oncogenesis apparently can occur within this narrow time channel, thereby making the drugs excellent probes in studies in chemical carcinogenesis. Although near ultraviolet light alone may transform the cells (8 to 16 min of irradiation), under the conditions studied ultraviolet light was neither toxic nor transforming.

Affinity Labels↗